1. Field of the Invention
The field of this invention is separable compositions for use in multiplexed detection.
2. Background of the Invention
As the human genome is elucidated, there will be numerous opportunities for performing assays to determine the presence of specific sequences, distinguishing between alleles in homozygotes and heterozygotes, determining the presence of mutations, evaluating cellular expression patterns, etc. In many of these cases one will wish to determine in a single reaction, a number of different characteristics of the same sample. Also, there will be an interest in determining the presence of one or more pathogens, their antibiotic resistance genes, genetic subtype and the like.
In many assays, there will be an interest in determining the presence of specific sequences, whether genomic, synthetic or cDNA. These sequences may be associated particularly with genes, regulatory sequences, repeats, multimeric regions, expression patterns, and the like
There is and will continue to be comparisons of the sequences of different individuals. It is believed that there will be about one polymorphism per 1,000 bases, so that one may anticipate that there will be an extensive number of differences between individuals. By single nucleotide polymorphism (snp""s) is intended that there will be a prevalent nucleotide at the site, with one or more of the remaining bases being present in substantially smaller percent of the population.
For the most part, the snp""s will be in non-coding regions, primarily between genes, but will also be present in exons and introns. In addition, the great proportion of the snp""s will not affect the phenotype of the individual, but will clearly affect the genotype. The snp""s have a number of properties of interest. Since the snp""s will be inherited, individual snp""s and/or snp patterns may be related to genetic defects, such as deletions, insertions and mutations involving one or more bases in genes. Rather than isolating and sequencing the target gene, it will be sufficient to identify the snp""s involved.
In addition, the snp""s may be used in forensic medicine to identify individuals. While other genetic markers are available, the large number of snp""s and their extensive distribution in the chromosomes, make the snp""s an attractive target. Also, by determining a plurality of snp""s associated with a specific phenotype, one may use the snp pattern as an indication of the phenotype, rather than requiring a determination of the genes associated with the phenotype.
The need to determine many analytes or nucleic acid sequences (for example multiple pathogens or multiple genes or multiple genetic variants) in blood or other biological fluids has become increasingly apparent in many branches of medicine. The need to study differential expression of multiple genes to determine toxicologically-relevant outcomes or the need to screen transfused blood for viral contaminants with high sensitivity is clearly evident.
Thus most multi-analyte assays or assays which detect multiple nucleic acid sequences involve multiple steps, have poor sensitivity and poor dynamic range (2 to 100-fold differences in concentration of the analytes is determined) and some require sophisticated instrumentation.
Some of the known classical methods for multianalyte assays include the following:
a. The use of two different radioisotope labels to distinguish two different analytes.
b. The use of two or more different fluorescent labels to distinguish two or more analytes.
c. The use of lanthanide chelates where both lifetime and wavelength are used to distinguish two or more analytes.
d. The use of fluorescent and chemiluminescent labels to distinguish two or more analytes.
e. The use of two different enzymes to distinguish two or more analytes.
f. The use of enzyme and acridinium esters to distinguish two or more analytes.
g. Spatial resolution of different analytes, for example, on arrays to identify and quantify multiple analytes.
h. The use of acridinium ester labels where lifetime or dioxetane formation is used to quantify two different viral targets.
Thus an assay that has higher sensitivity, large dynamic range (103 to 104-fold differences in target levels), greater degree of multiplexing, and fewer and more stable reagents would increase the simplicity and reliability of multianalyte assays.
The need to identify and quantify a large number of bases or sequences potentially distributed over centimorgans of DNA offers a major challenge. Any method should be accurate, reasonably economical in limiting the amount of reagents required and providing for a single assay, which allows for differentiation of the different snp""s or differentiation and quantitation of multiple genes.
Finally, while nucleic acid sequences provide extreme diversity for situations that may be of biological or other interest, there are other types of compounds, such as proteins in proteomics that may also offer opportunities for multiplexed determinations.
Holland (Proc. Natl. Acad. Sci. USA (1991) 88:7276) discloses the exonuclease activity of the thermostable enzyme Thermus aquaticus DNA polymerase in PCR amplification to generate specific detectable signal concomitantly with amplification.
The TaqMan assay is discussed by Lee in Nucleic Acid Research (1993) 21:16 3761).
White (Trends Biotechnology (1996) 14(12):478-483) discusses the problems of multiplexing in the TaqMan(copyright) assay.
Marino, Electrophoresis (1996) 17:1499 describes low-stringency-sequence specific PCR (LSSP-PCR). A PCR amplified sequence is subjected to single primer amplification under conditions of low stringency to produce a range of different length amplicons. Different patterns are obtained when there are differences in sequence. The patterns are unique to an individual and of possible value for identity testing.
Single strand conformational polymorphism (SSCP) yields similar results. In this method the PCR amplified DNA is denatured and sequence dependent conformations of the single strands are detected by their differing rates of migration during gel electrophoresis. As with LSSP-PCR above, different patterns are obtained that signal differences in sequence. However, neither LSSP-PCR nor SSCP gives specific sequence information and both depend on the questionable assumption that any base that is changed in a sequence will give rise to a conformational change that can be detected. Pastinen, Clin. Chem. (1996) 42:1391 amplifies the target DNA and immobilizes the amplicons. Multiple primers are then allowed to hybridize to sites 3xe2x80x2 and contiguous to a snp (xe2x80x9csingle nucleotide polymorphismxe2x80x9d) site of interest. Each primer has a different size that serves as a code. The hybridized primers are extended by one base using a fluorescently labeled dideoxynucleoside triphosphate. The size of each of the fluorescent products that is produced, determined by gel electrophoresis, indicates the sequence and, thus, the location of the snp. The identity of the base at the snp site is defined by the triphosphate that is used. A similar approach is taken by Haff, Nucleic Acids Res. (1997) 25:3749 except that the sizing is carried out by mass spectroscopy and thus avoids the need for a label. However, both methods have the serious limitation that screening for a large number of sites will require large, very pure primers that can have troublesome secondary structures and be very expensive to synthesize.
Hacia, Nat. Genet. (1996) 14:441 uses a high-density array of oligonucleotides. Labeled DNA samples are allowed to bind to 96,600 20-base oligonucleotides and the binding patterns produced from different individuals were compared. The method is attractive in that SNP""s can be directly identified, but the cost of the arrays is high and non-specific hybridization may confound the accuracy of the genetic information.
Fan (Oct. 6-8, 1997, IBC, Annapolis Md.) has reported results of a large scale screening of human sequence-tagged sites. The accuracy of single nucleotide polymorphism screening was determined by conventional ABI resequencing.
Allele specific oligonucleotide hybridization along with mass spectroscopy has been discussed by Ross in Anal. Chem. (1997) 69:4197.
Holland, et al., PNAS USA (1991) 88, 7276-7280, describes use of DNA polymerase 5xe2x80x2-3xe2x80x2 exonuclease activity for detection of PCR products.
U.S. Pat. No. 5,807,682 describes probe compositions for detecting a plurality of nucleic acid targets.
Compounds and methods are provided for multiplexed determinations affording convenient separation of released identifying tags based on individual physical, properties of the tags. The methods can be performed in a single vessel and may involve a plurality of reagents added simultaneously or consecutively. In one group of embodiments, mass will be involved in the characteristic allowing for separation. One group of identifying tags for electrokinetic analysis is characterized by having regions, which serve as (1) a cleavable linking region; (2) a mass-modifying region; (3) a charge-modifying region: and (4) a detectable region, the number of different regions depending in part on the method of separation and identification. Compounds that have these distinctive regions find use in conjunction with other compounds where the regions are combined in the same moiety. Of particular interest is the use of building blocks for forming the compounds, where the synthesis is performed in a repetitive manner using the same linking chemistry at a plurality of stages. The subject compounds are linked to binding compounds for identification to provide identifying reagents, where binding of an identifying reagent target in an assay system results in the release of the identifying tag (hereinafter referred to as an xe2x80x9ceTag(trademark) reporterxe2x80x9d) where the eTag reporters can be differentiated. Large numbers of eTag reporters can be provided in kits comprising a linking functionality for bonding to the binding compounds or kits of building blocks can be provided for synthesizing eTag reporters in situ in conjunction with the synthesis of the binding compound. Of particular interest is the use of the subject eTag reporters in identification of nucleic acids and proteins.